PROJECT SUMMARY Human tumor cells, including breast cancers, exhibit aberrant DNA double-strand break (DSB) repair pathways, which drive genomic instability and carcinogenesis, and most importantly, may serve as novel molecular targets that can be used to reverse treatment resistance phenotypes. However, the precise mechanisms governing the repair of DSBs and how their dysregulation, including upstream signaling in cancers, contributes to tumor cell resistance remains unclear. DSBs are induced by many types of cancer treatments, including ionizing radiation (IR), PARP inhibitors, and chemotherapy. As such, the overarching goal of this proposal is to determine the critical processes by which tumor cells respond to DSBs, as directed by SAMHD1, and how these repair pathways can be exploited to improve breast cancer treatment. SAMHD1 is recognized for its dNTP triphosphohydrolase activity, which restricts HIV-1 and other viral infections and for mutations associated with Aicardi Goutires syndrome (AGS), an autoimmune disorder. SAMHD1 is also overexpressed in 27% of breast cancers. We were the first to identify a novel role for SAMHD1 in promoting the end resection step of homologous recombination (HR) repair independent of its dNTPase activity, which has since been independently validated. Our preliminary data indicate that high SAMHD1 expression is associated with poor survival in breast cancer patients treated with adjuvant IR, suggesting that SAMHD1 overexpression contributes to IR resistance. Using a proteomic approach, we found that SAMHD1 interacts with a number of DNA damage response (DDR) proteins, including CtIP and SIRT1. Our data suggest that SAMHD1 recruits CtIP to DSBs to facilitate DNA end resection and HR independent of its dNTPase activity, and moreover, that SIRT1 directs SAMHD1 function in DSB repair through deacetylation. Finally, we developed a novel therapeutic strategy whereby targeting SAMHD1 for degradation with Vpx, a lentivirus accessory protein, packaged in virus-like particles (VLPs), sensitizes breast cancer cells to DSB-inducing agents. Thus, we hypothesize that in addition to its role in dNTP metabolism, SAMHD1 responds to SIRT1-mediated deacetylation to maintain genome integrity and govern breast cancer treatment resistance by non-catalytically directing DNA end resection and HR through CtIP, which may be exploited to improve breast cancer control. Using innovative genetic, cell biological, biochemical, structural, and in vivo approaches, we propose the following specific aims: 1) Determine the role of SAMHD1 in directing CtIP in DSB repair; 2) Determine the regulation of SAMHD1 in DSB repair by SIRT1; 3) Establish SAMHD1 as a potentially new molecular target for cancer therapy. Completion of this work will define how SAMHD1 non- catalytically directs CtIP function in DSB repair to maintain genome integrity and govern breast cancer treatment resistance, connect the SIRT1 acetylome with SAMHD1-regulated DSB repair, and establish proof of concept for the use of VLPs containing Vpx to target SAMHD1 as a novel therapeutic approach for improving breast cancer control.